Interaction between silver fir (Abies alba) wood water extract and lactobacilli.

Water extract from silver fir (Abies alba) wood represents a rich source of lignans and other phenols that are effective in different pathological conditions, such as diabetes, cardiovascular diseases and psoriasis. Its interaction with the gastrointestinal environment is crucial when the extract is orally administered. In this study we tested the in-vitro interaction between water extract of silver fir wood and ten different Lactobacillus species that are found in the gastrointestinal tract, vagina or are used in food industry. We tested both ways of interaction: 1) the bacterial influence on the chemical composition of the extract and 2) influence of the extract on the bacterial growth. We demonstrated that the extract is compatible with all of the bacteria and does not impair their growth. Furthermore the extract acted as a prebiotic for some bacteria including: L. paracasei, L. acidophilus, L. rhamnosus, L. gasseri, L. crispatus and L. bulgaricus, suggesting that the compounds in the extract can stimulate their growth. However, the ten lactobacilli did not show any chemical changes in lignan metabolism and the production of enterodiol and enterolactone, which are considered the final metabolic products of lignans and are produced by different gut bacteria. This study indicates that the silver fir wood extract is nutritious for some Lactobacillus bacteria and can be used as a prebiotic.

Efficacy of a polyphenolic extract from silver fir (Abies alba) bark on psoriasis: a randomised, double-blind, placebo-controlled trial.

Silver fir (Abies alba) bark extract contains a mixture of bioactive polyphenols. We tested their effectiveness in the treatment of psoriasis in order to further investigate the potential topical anti-inflammatory activity of polyphenols by means of a randomized, double-blind, placebo-controlled add-on clinical trial, after having examined their ability to downregulate the expression of IL-1ß cytokine in monocyte/macrophage primary cell culture. 61 patients with mild psoriasis met the inclusion criteria and were willing to comply with protocol requirements, were enrolled in the study. The severity of the disease was measured by psoriasis area severity index (PASI). Treatment efficacy was evaluated by assessing erythema (E, 0 to 4-point scale), desquamation (D, 0 to 4-point scale) and induration (I, 0 to 4-point scale) of lesions before and after the treatment. All patients enrolled in the study had symmetrical psoriasis plaques on the skin. All patients received O/V ointment with 2% of silver fir bark extract and/or placebo, respectively. We compared medications by right/left intra-patient comparison, so that the control group was always contralateral of the tested one. Location of the tested or control site was randomised, using a computer-generated randomisation schedule. Silver fir extract was well-tolerated. A superiority of active treatment above placebo, based on the clinical investigational PASI score system was observed by 15 % in all volunteers and in 40% regarding the improvement of psoriasis on elbows. However, statistical analysis showed no significant differences between placebo and active treatment with the extract from silver fir bark (p < 0.05).

Randomised, double blind, cross-over, placebo and active controlled human pharmacodynamic study on the influence of silver fir wood extract (Belinal) on post-prandial glycemic response.

The aim of this study was to test the extract from silver fir wood (Belinal) on the reduction of the blood glucose concentrations after consumption of a standard meal. 31 healthy participants consumed 100 g of white bread 4 times (with 1 week washout period, consequently) concomitantly with a capsule of Belinal, capsule of chestnut wood extract, placebo or acarbose (active control). Glucose and insulin in the blood were measured before and after the meal. The area under the curve of glucose concentration in blood after the meal was 35 % lower when Belinal was added compared with the placebo group (p = 0.019). Acarbose lowered the area for 43 % (p = 0.002). By this, we proved that the effect of Belinal might be beneficial for prevention of diabetes. This is the first study that provides a scientific rationale for use of silver fir wood extract as food supplement for reduction of health risks connected to type 2 diabetes mellitus.

Does ertepenem alter the susceptibility of Pseudomonas aeruginosa to carbapenems?

We investigated the effect of ertapenem on carbapenem susceptibility of Pseudomonas aeruginosa. Antibiotic consumption was recorded monthly in defined daily doses (DDD)/100 patient-days in the infectious diseases (ID), abdominal surgery (AS), and surgical intensive care units (SICU) of a teaching hospital from January 2005 to December 2008. Trends of decreased susceptibility of P. aeruginosa were observed in all three units. After the introduction of ertapenem, the number of P. aeruginosa isolates/ 1000 patients-days per month increased in AS and in SICU (p=0.05). The increase in carbapenem non-susceptible isolates/1000 patients-days in the same units was less significant (p=0.07 and p=0.054). Correlations between ertapenem and the carbapenem non-susceptibility for the lagtime of 1 to 6 months ahead gave no significant result. In the SICU, 30% of variability of carbapenem non-susceptibility could be predicted by the consumption of ertapenem. There is no evidence that ertapenem alters the P. aeruginosa susceptibility to carbapenems, but the relationship deserves further observation.

The impact of total control of antibiotic prescribing by infectious disease specialist on antibiotic consumption and cost.

We analyzed the impact on antimicrobial consumption of antimicrobial therapy totally guided by an infectious disease specialist (iD). in a teaching hospital antibiotic consumption was recorded yearly from 1998 to 2005 in three units. Antibiotic policy was introduced in 1999. in Unit A, restricted antimicrobials were prescribed after approval by the head of the unit. the iD was involved as a consultant upon request. in unit b, restricted antimicrobials were approved by an iD. All other antimicrobials were prescribed by the physicians on the ward. in unit C, all antimicrobials were prescribed by an iD. A significant decreasing trend in antibiotic consumption in defined daily doses (DDD) per patient day and per admission, and cost of antimicrobials per patient was observed in unit C, a decreasing trend in antibiotic consumption per patient was observed in unit b as well. totally iD guided antimicrobial therapy is an efficient method of antimicrobial policy.

Molecular genetics of the ubiquitin-proteasome system: lessons from yeast.

Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATalpha2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to alpha2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as alpha2. The 26S proteasome is responsible for alpha2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.

Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies.

Interference with fat hydrolysis results in the reduced use of ingested lipids. Inhibition of pancreatic lipase reduces the efficiency of fat absorption in the small intestine and thereby initiates modest long-term reduction in body weight. In an attempt to select peptides with affinity for the surface of pancreatic lipase and potential inhibitory activity, a random, cyclic heptapeptide phage-displayed library was used. Five independent selections, differing in elution step, were performed. In three selection protocols, a sequential elution strategy was applied in anticipation of improving the selection of high-affinity clones. Four heptapeptides with the highest affinity, seemingly for pancreatic lipase, were selected, synthesized, and characterized for their capacity to inhibit enzyme function. Although no clear consensus among the sequenced peptides was found, one of the selected peptides inhibited pancreatic lipase with an apparent inhibition constant of 16 muM.

Aetiology and clinical presentation of mild community-acquired bacterial pneumonia.

A prospective study was initiated to analyse the bacterial aetiology and clinical picture of mild community-acquired pneumonia in Slovenia using the previously described Pneumonia Severity Index. Radiographically confirmed cases of pneumonia in patients treated with oral antibiotics in seven study centres were included. An aetiological diagnosis was attempted using culture of blood and sputum, urinary antigen testing for Streptococcus pneumoniae and Legionella pneumophila, and antibody testing for Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila in paired serum samples. One hundred thirteen patients were evaluable for clinical presentation and 109 for aetiological diagnosis. At least one pathogen was detected in 62.4% patients. The most common causative agents were Mycoplasma pneumoniae in 24.8%, Chlamydia pneumoniae in 21.1%, and Streptococcus pneumoniae in 13.8% of patients. Dual infection was detected in 8.3% of patients. Most patients suffered from cough, fatigue, and fever. Patients with atypical aetiology of pneumonia differed from those with typical bacterial pneumonia or pneumonia of unknown aetiology in age, presence of dyspnea, and bronchial breathing on lung auscultation. Patients with pneumococcal, chlamydial, and mycoplasmal infections differed in age, risk class, presence of dyspnea, bronchial breathing, and proteinuria. There was an overlap of other clinical symptoms, underlying conditions, and laboratory and radiographic findings among the groups of patients classified by aetiology. Since patients with mild community-acquired pneumonia exhibit similar clinical characteristics and, moreover, since a substantial proportion of cases are attributable to atypical bacteria, broad-spectrum antibiotic treatment seems to be recommended.

Oxidation of lovastatin in the solid state and its stabilization with natural antioxidants.

Oxidative instability of the hydroxy methylglutaryl Co-A reductase inhibitor lovastatin in the solid state and stabilization with natural antioxidants (ascorbic acid, rutin, gallic acid, quercetin and caffeic acid) was investigated. Lovastatin in the solid state and binary mixtures with 10% (w/w), 25% (n/n), 12.5% (n/n) and 6.3% (n/n) of each of the antioxidants were prepared. Oxidation experiments were performed on the scanning calorimeter using dynamic oxygen atmosphere. The amount of non-oxidized lovastatin was determined using HPLC. The results of the experiments have shown that lovastatin is unstable to oxidation under higher temperatures and in the presence of oxygen, and that some antioxidants markedly stabilize the drug. The most significant antioxidative effect was seen with caffeic acid and rutin, followed by gallic acid and quercetin. Ascorbic acid was only moderately effective. The results prove that flavonoids do have significant antioxidative potential. This phenomenon can be used to improve oxidative stability of drugs such as lovastatin which are sensitive to the presence of oxygen.

Reversed-polarity capillary zone electrophoretic analysis of usnic acid.

A capillary zone electrophoretic (CZE) method for the determination of usnic acid is described for the first time. Usnic acid is an antibiotic substance from lichens. Due to its low solubility in water, a high content of methanol in CZE buffer is required. Because of the methanol in the buffer, the electroosmotic flow velocity was lower than the electrophoretic mobility of usnic acid. Accordingly, the use of reversed-polarity (with the anode on the detector side of the capillary) was necessary. The optimal buffer composition was 50 mM NaOH, 20 mM acetic acid and 5% water in methanol. The detection limit of UV detector at 290 nm for usnic acid in the injected extract was 3.5 mg/L and the relative standard deviation of the normalized peak area was 3.3% at 250 mg/L.

Screening for selective thrombin inhibitors in mushrooms.

Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. A number of pharmacologically active secondary metabolites from mushrooms have already been isolated, thus providing the rationale for screening for new thrombin inhibitors in mushrooms. In this study, inhibitory activities of mushroom extracts on thrombin and trypsin were measured using the chromogenic substrates H-D-phenylalanine-L-pipecolyl-L-arginine-paranitroaniline dihydrochloride (S-2238) for thrombin and N-benzoyl-D,L-Arg-p-nitroanilide (BAPNA) for trypsin. The inhibitory activities of extracts from 95 Basidiomycete species have been determined. The majority of samples inhibited trypsin and thrombin with various potencies; however, some extracts showed no activity against one or both of the enzymes. An aqueous extract of Gleophyllum odoratum exhibited high inhibitory activity on both thrombin and trypsin (72 and 60%, respectively), while extracts of Clitocybe gibba, Amanita virosa, Cantharellus lutescens, Suillus tridentinus, Hypoloma fasciculare and Lactarius badiosanguineus considerably inhibited thrombin (49, 48, 36, 34, 32 and 31%, respectively) and showed no inhibitory activity on trypsin. The results at this point are promising for further research with the objective of finding an effective and safe thrombin inhibitor.

Screening of selected plant extracts for in vitro inhibitory activity on HIV-1 reverse transcriptase (HIV-1 RT).

Methanolic-aqueous extracts of 70 plants were investigated for their ability to inhibit HIV-1 reverse transcriptase activity in vitro. Two thirds of the extracts screened showed more than 50% inhibition. Two extracts inhibited the enzyme completely while four exhibited more than 90% inhibition. Tannins as nonspecific HIV-1 RT inhibitors were detected and removed from the extracts. The IC50 values of the most potent extracts after the removal of tannins for the HIV-1 RT inhibition are as follows: Sambucus racemosa 0.017 mg/ml and Geranium phaeum 0.067 mg/ml. Daunomycine was chosen as a standard substance in the non-radioactive immuno assay used for screening. As a result from the future isolation and characterization of these compounds, new leading structures are expectable.

Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Comparison to the radioactive technique.

A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.

Extraction of rutin from buckwheat (Fagopyrum esculentumMoench) seeds and determination by capillary electrophoresis.

The content of the flavonoid rutin was determined in different milling fractions of buckwheat seeds and in buckwheat stems, leaves, and flowers. The extraction was performed by using a solvent containing 60% of ethanol and 5% of ammonia in water. The extracts were analyzed by capillary electrophoresis (running buffer of 50 mM borate (pH 9.3), 100 mM sodium dodecyl sulfate; determination at 380 nm). In bran fractions the concentration of rutin was 131-476 ppm, and in flour fractions 19-168 ppm. On average, about 300, 1000, and 46000 ppm of rutin were found in leaves, stems, and flowers, respectively. The results indicate that buckwheat could be an important nutritional source of flavonoids, especially in countries with a low mean daily flavonoid intake.

Quantitative phytochemical analyses of six hypericum species growing in slovenia.

The 74 samples of six HYPERICUM species (H. PERFOROTUM, H. HIRSUTUM, H. MACULATUM, H. TETRAPTERUM, H. MONTANUM, AND H. HUMIFUSUM) were collected around Slovenia and analysed for the content of ten substances (rutin, hyperoside, isoquercetin, quercitrin, quercetin, I3,II8-biapigenin, amentoflavone, pseudohypericin, hypericin, and hyperforin). The flowers were analysed separately from the green parts of the plants (herbs). The highest content of most of the substances was found in the flowers of H. PERFOROTUM. Among the herbal samples (without flowers), H. MONTANUM and H. HIRSUTUM contained significantly higher levels of amentoflavone (average 3-fold and 2.5-fold higher, respectively), than the herbs of H. PERFOROTUM. In the herbal part of H. PERFOROTUM the contents of all constituents strongly correlate with the contents of the same compound in flowers, except for the content of amentoflavone, which is independent in these two parts. Rutin and hyperoside are in positive correlation, and quercitrin is in negative correlation with the altitude of the growing site.

Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30-40% of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60% decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid-treated muscle.

Jasmonic acid inducible aspartic proteinase inhibitors from potato.

A new cDNA clone coding for an aspartic proteinase inhibitor homologue was isolated from a potato tuber cDNA library. Southern blot analysis was used to study the structural diversity of the aspartic proteinase inhibitor gene family in several species of the Solanaceae. The existence of sequence-homologous genes was confirmed in the genomic DNA of different potato cultivars (Solanum tuberosum L. cv. Désirée, Pentland Squire and Igor), tomato (Lycopersicon esculentum Mill.), aubergine (S. melongena L.) and a wild type of bittersweet (S. dulcamara L.). Northern blot hybridization of total RNA, isolated from leaves under non-stress conditions, of different solanaceous species and of potato tubers showed that the gene transcripts encoding aspartic proteinase inhibitors occur mainly in potato tubers. The presence of several cathepsin D inhibitor isoforms has been detected at the protein level. At least four isoforms were isolated by affinity chromatography on cathepsin D-Sepharose and characterized. Additionally, exogenous treatment of potato plantlets by jasmonic acid (JA) over a wide range of concentrations (0-100 microM) was performed in a stem node culture in vitro. We demonstrated that the expression of aspartic proteinase inhibitor mRNA was drastically induced in potato shoots at concentrations of 50-100 microM JA.

Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat; comparison to the radioactive technique.

In this preliminary report nonradioactive digoxigenine-based and radioactive in situ hybridization procedures for the localization of acetylcholinesterase mRNA were tested and compared in rat brain. General patterns of Ache mRNA localization observed by both techniques did not differ significantly and were practically the same as reported in previous in situ studies on the mammalian brain. Shorter procedure time and avoidance of precautions necessary at work with radioactive materials are major advantages of nonradioactive technique. Under- and over- staining can be prevented by direct examination of coloring reaction. Faint staining in the control experiment with heterologous DNA suggests that proper stringency is essential for the specificity of staining.